Quantitation of DNA for Forensic DNA Typing by qPCR (quantitative PCR): Singleplex and Multiplex Modes for Nuclear and Mitochondrial Genomes, and the Y Chromosome

摘要

Several new qPCR assays have been developed in single and multiplex modes for quantifying DNA in forensic samples. These assays, listed below, were developed to improve the accuracy and precision of DNA quantifications, as well as to provide tools to increase the efficiency and throughput of the analysis of complex forensic samples: (i) a duplex assay that quantifies simultaneously the human nuclear and mitochondrial genomes. This assay was developed to quantify DNA in the degraded samples that are often encountered in missing persons cases. It has been optimized and validated for forensic casework. (ii) a triplex assay that quantifies two human nuclear target sequences, one target that is approximately 170-190 basepairs (bp) in length and the other 67 bp in length, and that also includes an internal positive control (IPC) assay. By comparing quantifications from the two differently sized nuclear target sequences, the assay effectively estimates the extent of DNA degradation in a sample. Moreover, the IPC portion of the assay provides a means for detecting for the presence of co-extracted PCR inhibitors. In addition to the triplex assay, a duplex assay, which quantifies the long nuclear target sequence and detects the IPC, has also been developed. (iii) several singleplex assays to quantify specifically the human Y-chromosome. All assays (i-iii) were developed utilizing 5-hydrolysis (TaqMan) detection chemistries for data collection on the ABI 7000 instrument. Most features should be portable to other real-time qPCR instruments capable of multiplex detection and so should be of general utility to the forensic community. Two of the assays ((i) and (ii)) have been described in manuscripts accepted for publication. We have also initialized formalized training in the application of qPCR to forensic samples through a course that is offered through the California Criminalistics Institute. This course includes discussion of the theory of qPCR, as well as hands-on laboratory and data analysis experiences with real-time qPCR assays developed here and elsewhere.